DOCSLIB.ORG

Isolation and Characterisation of a Cathepsin L Proteinase

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Isolation and Characterisation of a Cathepsin L Proteinase Fasciola hepatica: Isolation and characterisation of a cathepsin L proteinase. Thesis Presented for the Degree of DOCTOR OF PHILOSOPHY by Angela M. Smith, B.Sc. under the supervision of John P. Dalton, Ph.D. School of Biological Sciences Dublin City University. I hereby certify that this material, which I now submit for assessment on the programme of study leading to the award of Ph.D. is entirely my own work and has not been taken from the work of others save and to the extent that such work has been cited and acknowledged within the text of my work. Signed: l/v Date Angela 1VI. Smith Date:. ACKNOWLEDGEMENTS I would like to thank Dr. John Dalton for all he has done for me during the last four years. His help, guidance and enthusiasm were much appreciated. I would also like to thank Dr. Paul Brindley for his help and advice during my six month visit to his laboratory in Australia, Dr. Alan Trudgett and his colleagues in Queen's University Belfast for their collaboration in the immunolocalisation studies, and Dr. Carlos Carmona of the Universidad de la República, Montevideo, Uruguay for his contribution to the antibody inhibition studies during his visit to our laboratory in Dublin. I would also like to express my gratitude to my parents for giving me so many opportunities, and to all my friends for their support and encouragement, especially during the last six months. Finally, I wish to thank the members of the Parasitology lab. in D. C. U. for the good times and the never boring lab excursions! To my parents. CONTENTS Abstract 1 Abbreviations 2 1.0 Introduction 4 2.0 Materials and Methods 36 2.1 Materials 37 2.2 Methods 41 2.2.1 Preparation of in vitro-released products from adult Fasciola hepatica 41 2.2.2 Sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) 41 2.2.3 Gelatin-substrate gel analysis of fluke in vitro released products 42 2.2.4 Protein estimation 42 2.2.5 HPLC analysis of E/S products 43 2.2.6 Assay for lgG2a cleaving activity 43 2.2.7 Proteinase assays with synthetic fluorogenic peptide substrates 43 2.2.8 Fluorogenic visualisation of proteinases in SDS-PAGE 44 2.2.9 Inhibition studies using diethylpyrocarbonate (DPC) and Z-F-A-CHN2 44 2.2.10 Purification of F. hepatica\gG cleaving cysteine proteinase 45 2.2.11 N-terminal sequence determination 46 2.2.12 Production of a polyclonal antiserum 46 2.2.13 Immunoblotting 47 2.2.14 Immunolocalisation studies 47 2.2.15 Inhibition of proteinase activity using anti- cathepsin L-like proteinase antibody 48 2.2.16 RNA isolation from adult F. hepatica worms 49 2.2.17 mRNA isolation 50 2.2.18 cDNA preparation 51 2.2.19 Construction of oligonucleotide primers 53 2.2.20 Polymerase chain reaction (PCR) 55 2.2.21 Subcloning of PCR gene fragments 55 2.2.22 Screening of recombinant colonies 56 2.2.23 Sequencing of subcloned PCR gene fragments 57 3.0 Results 58 3.1 Characterisation of IgG cleaving enzyme in adult fluke E/S products 59 3.1.1 Demonstration of IgG cleavage 59 3.1.2 HPLC analysis of E/S products 61 3.1.3 Direct visualisation of proteinases in HPLC fractions 61 3.2 Inhibition studies with DPC and Z-F-A-CHN2 64 3.2.1 Inhibition of the active site histidine residue with DPC 64 3.2.2 Inhibition with Z-F-A-CHN2 67 3.3 Purification of the cathepsin L-like cysteine proteinase 67 3.4 N-terminal sequence determination 69 3.5 Immunoblotting studies 74 3.6 Light- and electron-microscope immunolocalisation studies 78 3.7 Inhibition of proteinase activity with anti-cathepsin L-like proteinase antibodies 81 3.7.1 Inhibition of GS-PAGE proteolytic activity 81 3.7.2 Inactivation of the IgG cleaving ability of the proteinase 84 3.7.3 Antibody-mediated eosinophil attachment to juvenile flukes 84 3.8 Cloning and sequencing of PCR amplified cysteine proteinase gene fragments 85 3.8.1 PCR amplification of cysteine proteinase gene fragments 86 3.8.2 Subcloning and sequence analysis 86 4.0 Discussion 93 5.0 References 122 6.0 Appendix 147 ABSTRACT Fasciola hepatica, a parasitic trematode, is the causative agent of liver fluke disease. It has been shown previously, that both the migratory and adult worm stage of the parasite secrete multiple cysteine proteinases when they are cultured overnight (Dalton & Heffernan, 1989). In this study, one of these proteinases has been purified by standard chromatographic techniques. The purified enzyme was characterised as a cathepsin L-like proteinase using synthetic substrates, inhibition studies, N-terminal sequencing and immunolocalisation studies. This is the first cathepsin L-like proteinase to be identified in a parasitic trematode. This cathepsin L-like proteinase is capable of cleaving immunoglobulin molecules, and is able to protect newly excysted juveniles from destruction by immune-effector cells when it is included in an eosinophil adherence assay. Antibodies to the purified proteinase are able to neutralise its proteolytic activity in vitro. A partial gene fragment encoding the cathepsin L-like proteinase has been obtained using PCR and subcloning techniques. The cathepsin L-like proteinase is present in all stages of F. hepatica and, hence, is considered an ideal target molecule at which to design a vaccine and/or drug, for use in the control of this agriculturally important parasitic disease. 1 ABBREVIATIONS BCIP 5-bromo-5-chloro-3-indolyl phosphate Bisacrylamide N, A/“-Methylene bisacrylamide BSA Bovine serum albumin DMSO Dimethyl sulphoxide DPC Diethylpyrocarbonate DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid disodium salt E-64 fra/7s-epoxysuccmyl-L-leucylamido(4-guanidino) butane FCS Foetal calf serum FITC Fluorescein isothiocyanate Hepes N-[2-hydroxyethyl] piperazine-N’[2-ethane sulphonic acid] IPTG Isopropyl-B-thiogalactopyranoside NBT Nitro blue tétrazolium PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline PMSF Phenylmethylsulphonyl fluoride RPMI Roswell Park Memorial Institute SDS Sodium dodecyl sulphate TEMED N, N, N’, N’-tetramethylethylenediamine Tris tris-(hydroxymethyl)-methylamine (2-amino- hydroxylmethyl) propane-1,3-diol Z-F-A-CHN2 /V-benzyloxcarbonyl-L-phenylalanine-L-alanine- 2 diazomethylketone Z-F-R-AMC A/-benzyloxcarbonyl-L-phenylalanine-L-arginine-7- amino-4-methylcoumarîn.HCI Z-R-AMC /V-benzyloxcarbonyl-L-arginine-7-amino-4- methylcoumarin.HCI Z-R-R-AMC A/-benzyloxcarbonyl-L-arginine-L-arginine-7-amino- 4-methylcoumarin.HCI 3 CHAPTER ONE INTRODUCTION 4 1.0 INTRODUCTION In 1947, Professor Stoll drew attention to the worldwide presence of helminth parasites in his article “This wormy world”. Helminth parasites infected 70 % of the then world population of approximately 2 billion (Stoll, 1947). Since that time, the prevalence of helminth infections has kept pace with the growth of the world population. If the trend continues till the year 2100, a predicted world population of 7-15 billion would harbour 5-10 billion helminth infections, unless special control measures are undertaken (Crompton, 1987). The term “helminth” (derived from the Greek words helm ins or helminthos), literally means “worm”, zoologically speaking however, it has a more precise connotation and is currently restricted to members of the phyla Platyhelminthes, Nematoda and Acanthocephala (Smyth, 1976). The study of helminths is now regarded as being confined to the study of parasitic worms. Helminths typically parasitise vertebrates, although invertebrates act as intermediate hosts. The helminth diseases in man and domestic animals are caused by three groups of parasites belonging to the classes of trem atoda (flatworms), nematoda (roundworms), and cestoda (tapeworms), and are distributed throughout the world (Singh & Sharma, 1991). There are approximately 200 recognised helminth parasites of man. Table 1.1 lists the parasites which are most common in humans. For most helminth infections the relationship between between infection and disease is complex, and disease is not necessarily an automatic outcome of infection (Bundy etal., 1992). Only a small proportion of those individuals with heavy infections are likely to develop overt disease. There is a low mortality/high morbidity rate associated with helminth infections, so although 5 Table 1.1 Parasitic helminth infections which are common to man, an example of a causative agent of each infection, and the numbers infected. Data obtained from Hopkins, (1992). Parasite infection & example Millions infected Ascariasis (Ascaris lumbricoides) 1000 Hookworm (Ancylostoma duodenale) 900 Trichuriasis (Trichuris trichiura) 750 Schistosomiasis (Schistosoma mansoni) 250 Filariasis (Wuchereria bancrofti) 90 Taeniasis (Taenia saginata) 70 Onchocerciasis (Onchocerca volvulus) 30 Fascioliasis (Fasciola hepatica) 17 Trichinosis (Trichinella spiralis) 11 millions of people may be infected with helminths, relatively few will actually die as a result of infection, which seems to prevent well-focused investigation into their control and treatment (Parkhouse & Harrison, 1989). In fact it is estimated that at least one quarter of the worlds population is infected with helminthic parasites (Bundy, 1992), and about 150,000 die each year as a result of these infections (Bundy, 1990). One feature in the evolution of some animals is the increasing complexity of their alimentary, respiratory and circulatory systems. The development of such systems was, of course, advantageous to these evolving organisms, but it was not without some inherent disadvantages. As each new organ system evolved, 6 especially those containing cavities or surfaces, it presented a habitat for potential parasites. These cavity containing organs appeared especially in vertebrates and every part of the vertebrate body capable of supporting parasitic life has been invaded (Smyth, 1976). A majority of helminths use the gastrointestinal tract as their favourite niche; however some parasites may also invade musculature, the blood circulatory system, and other parts of the body such as lungs, liver, lymphatics, and eyes, producing serious clinical complications.
Load more

Recommended publications
  • Supplementary Data
    Supplementary Data for Quantitative Changes in the Mitochondrial Proteome from Subjects with Mild Cognitive Impairment, Early Stage and Late Stage Alzheimer’s disease Table 1 - 112 unique, non-redundant proteins identified and quantified in at least two of the three analytical replicates for all three disease stages. Table 2 - MCI mitochondrial samples, Protein Summary Table 3 - MCI mitochondrial samples, Experiment 1 Table 4 - MCI mitochondrial samples, Experiment 2 Table 5 - MCI mitochondrial samples, Experiment 3 Table 6 - EAD Mitochondrial Study, Protein Summary Table 7 - EAD Mitochondrial Study, Experiment 1 Table 8 - EAD Mitochondrial Study, Experiment 2 Table 9 - EAD Mitochondrial Study, Experiment 3 Table 10 - LAD Mitochondrial Study, Protein Summary Table 11 - LAD Mitochondrial Study, Experiment 1 Table 12 - LAD Mitochondrial Study, Experiment 2 Table 13 - LAD Mitochondrial Study, Experiment 3 Supplemental Table 1. 112 unique, non-redundant proteins identified and quantified in at least two of the three analytical replicates for all three disease stages. Description Data MCI EAD LAD AATM_HUMAN (P00505) Aspartate aminotransferase, mitochondrial precursor (EC Mean 1.43 1.70 1.31 2.6.1.1) (Transaminase A) (Glutamate oxaloacetate transaminase 2) [MASS=47475] SEM 0.07 0.09 0.09 Count 3.00 3.00 3.00 ACON_HUMAN (Q99798) Aconitate hydratase, mitochondrial precursor (EC 4.2.1.3) Mean 1.24 1.61 1.19 (Citrate hydro-lyase) (Aconitase) [MASS=85425] SEM 0.05 0.17 0.18 Count 3.00 2.00 3.00 ACPM_HUMAN (O14561) Acyl carrier protein, mitochondrial
    [Show full text]
  • Novel Binding Partners of PBF in Thyroid Tumourigenesis
    NOVEL BINDING PARTNERS OF PBF IN THYROID TUMOURIGENESIS By Neil Sharma A thesis presented to the College of Medical and Dental Sciences at the University of Birmingham for the Degree of Doctor of Philosophy Centre for Endocrinology, Diabetes and Metabolism, School of Clinical and Experimental Medicine August 2013 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. SUMMARY Thyroid cancer is the most common endocrine cancer, with a rising incidence. The proto-oncogene PBF is over-expressed in thyroid tumours, and the degree of over-expression is directly linked to patient survival. PBF causes transformation in vitro and tumourigenesis in vivo, with PBF-transgenic mice developing large, macro-follicular goitres, effects partly mediated by the internalisation and repression of the membrane-bound transporters NIS and MCT8. NIS repression leads to a reduction in iodide uptake, which may negatively affect the efficacy of radioiodine treatment, and therefore prognosis. Work within this thesis describes the use of tandem mass spectrometry to produce a list of potential binding partners of PBF. This will aid further research into the pathophysiology of PBF, not just in relation to thyroid cancer but also other malignancies.
    [Show full text]
  • Fasciola Hepatica
    Fasciola hepatica Robinson, M., Dalton, J., O'Brien, B., & Donnelly, S. (2013). Fasciola hepatica: The therapeutic potential of a worm secretome. International Journal for Parasitology, 43(3-4), 283–291. https://doi.org/10.1016/j.ijpara.2012.11.004 Published in: International Journal for Parasitology Document Version: Peer reviewed version Queen's University Belfast - Research Portal: Link to publication record in Queen's University Belfast Research Portal Publisher rights NOTICE: this is the author’s version of a work that was accepted for publication in International Journal for Parasitology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in International Journal for Parasitology, [VOL 43, ISSUE 2-3, 2013] General rights Copyright for the publications made accessible via the Queen's University Belfast Research Portal is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact [email protected].
    [Show full text]
  • Proteomics and Phylogenetic Analysis of the Cathepsin L
    Research Proteomics and Phylogenetic Analysis of the Cathepsin L Protease Family of the Helminth Pathogen Fasciola hepatica EXPANSION OF A REPERTOIRE OF VIRULENCE-ASSOCIATED FACTORS* Mark W. Robinson‡§¶, Jose F. Tort‡ʈ, Jonathan Lowther‡, Sheila M. Donnelly‡, Emily Wong‡, Weibo Xu‡, Colin M. Stack‡**, Matthew Padula‡‡, Ben Herbert‡‡, and John P. Dalton‡§§ Cathepsin L proteases secreted by the helminth pathogen zymes. The prosegment region was highly conserved Fasciola hepatica have functions in parasite virulence in- between the clades except at the boundary of prosegment cluding tissue invasion and suppression of host immune and mature enzyme. Despite the lack of conservation at this Downloaded from responses. Using proteomics methods alongside phylo- section, sites for exogenous cleavage by asparaginyl en- genetic studies we characterized the profile of cathepsin dopeptidases and a Leu-Ser2His motif for autocatalytic L proteases secreted by adult F. hepatica and hence iden- cleavage by cathepsin Ls were preserved. Molecular & tified those involved in host-pathogen interaction. Phylo- Cellular Proteomics 7:1111–1123, 2008. genetic analyses showed that the Fasciola cathepsin L gene family expanded by a series of gene duplications https://www.mcponline.org followed by divergence that gave rise to three clades associated with mature adult worms (Clades 1, 2, and 5) The helminth pathogens Fasciola hepatica and Fasciola and two clades specific to infective juvenile stages gigantica are the causative agents of liver fluke disease (fas- (Clades 3 and 4). Consistent with these observations our ciolosis) in sheep and cattle. Although infections of F. hepat- proteomics studies identified representatives from ica occur predominantly in regions with temperate climates, Clades 1, 2, and 5 but not from Clades 3 and 4 in adult F.
    [Show full text]
  • Spatiotemporal Proteomics Uncovers Cathepsin-Dependent Host Cell
    bioRxiv preprint doi: https://doi.org/10.1101/455048; this version posted November 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 1 Spatiotemporal proteomics uncovers cathepsin-dependent host 2 cell death during bacterial infection 3 Joel Selkrig1, Nan Li1,2,3, Jacob Bobonis1,4, Annika Hausmann5, Anna Sueki1,4, Haruna 4 Imamura6, Bachir El Debs1, Gianluca Sigismondo3, Bogdan I. Florea7, Herman S. 5 Overkleeft7, Pedro Beltrao6, Wolf-Dietrich Hardt5, Jeroen Krijgsveld3‡, Athanasios Typas1‡. 6 7 1 European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Meyerhofstrasse 1, 69117 8 Heidelberg, Germany. 9 2 Institute of Synthetic Biology (iSynBio), Shenzhen Institutes of Advanced Technology (SIAT), 10 Chinese Academy of Sciences (CAS), 1068 Xueyuan Avenue, Shenzhen University Town, Shenzhen, 11 China. 12 3 German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany. 13 4 Collaboration for joint PhD degree between EMBL and Heidelberg University, Faculty of Biosciences 14 5 Institute of Microbiology, ETH Zurich, CH-8093 Zurich, Switzerland. 15 6 European Bioinformatics Institute, European Molecular Biology Laboratory, Wellcome Trust Genome 16 Campus, Hinxton, Cambridge, CB10 1SD 17 7 Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, Einsteinweg 18 55, 2333 CC Leiden, the Netherlands. 19 20 21 ‡ to whom correspondence should be addressed: 22 [email protected] & [email protected] 23 24 SUMMARY 25 Immune cells need to swiftly and effectively respond to invading pathogens.
    [Show full text]
  • Data Sheet Cathepsin V Inhibitor Screening Assay Kit Catalog #79589 Size: 96 Reactions
    6405 Mira Mesa Blvd Ste. 100 San Diego, CA 92121 Tel: 1.858.202.1401 Fax: 1.858.481.8694 Email: [email protected] Data Sheet Cathepsin V Inhibitor Screening Assay Kit Catalog #79589 Size: 96 reactions BACKGROUND: Cathepsin V, also called Cathepsin L2, is a lysosomal cysteine endopeptidase with high sequence homology to cathepsin L and other members of the papain superfamily of cysteine proteinases. Its expression is regulated in a tissue-specific manner and is high in thymus, testis and cornea. Expression analysis of cathepsin V in human tumors revealed widespread expression in colorectal and breast carcinomas, suggesting a possible role in tumor processes. DESCRIPTION: The Cathepsin V Inhibitor Screening Assay Kit is designed to measure the protease activity of Cathepsin V for screening and profiling applications. The Cathepsin V assay kit comes in a convenient 96-well format, with purified Cathepsin V, its fluorogenic substrate, and Cathepsin buffer for 100 enzyme reactions. In addition, the kit includes the cathepsin inhibitor E- 64 for use as a control inhibitor. COMPONENTS: Catalog # Component Amount Storage 80009 Cathepsin V 10 µg -80 °C Fluorogenic Cathepsin Avoid 80349 Substrate 1 (5 mM) 10 µl -20 °C multiple *4X Cathepsin buffer 2 ml -20 °C freeze/thaw E-64 (1 mM) 10 µl -20 °C cycles! 96-well black microplate * Add 120 µl of 0.5 M DTT before use. MATERIALS OR INSTRUMENTS REQUIRED BUT NOT SUPPLIED: 0.5 M DTT in aqueous solution Adjustable micropipettor and sterile tips Fluorescent microplate reader APPLICATIONS: Great for studying enzyme kinetics and screening small molecular inhibitors for drug discovery and HTS applications.
    [Show full text]
  • Felipe Jun Fuzita Molecular Physiology of Digestion In
    FELIPE JUN FUZITA MOLECULAR PHYSIOLOGY OF DIGESTION IN ARACHNIDA: FUNCTIONAL AND COMPARATIVE-EVOLUTIONARY APPROACHES Thesis presented to the Programa de Pós-Graduação Interunidades em Biotecnologia USP/Instituto Butantan/IPT, to obtain the Title of Doctor in Biotechnology. São Paulo 2014 FELIPE JUN FUZITA MOLECULAR PHYSIOLOGY OF DIGESTION IN ARACHNIDA: FUNCTIONAL AND COMPARATIVE-EVOLUTIONARY APPROACHES Thesis presented to the Programa de Pós-Graduação Interunidades em Biotecnologia USP/Instituto Butantan/IPT, to obtain the Title of Doctor in Biotechnology. Concentration area: Biotechnology Advisor: Dr. Adriana Rios Lopes Rocha Corrected version. The original electronic version is available either in the library of the Institute of Biomedical Sciences and in the Digital Library of Theses and Dissertations of the University of Sao Paulo (BDTD). São Paulo 2014 DADOS DE CATALOGAÇÃO NA PUBLICAÇÃO (CIP) Serviço de Biblioteca e Informação Biomédica do Instituto de Ciências Biomédicas da Universidade de São Paulo © reprodução total Fuzita, Felipe Jun. Molecular physiology of digestion in Arachnida: functional and comparative-evolutionary approaches / Felipe Jun Fuzita. -- São Paulo, 2014. Orientador: Profa. Dra. Adriana Rios Lopes Rocha. Tese (Doutorado) – Universidade de São Paulo. Instituto de Ciências Biomédicas. Programa de Pós-Graduação Interunidades em Biotecnologia USP/IPT/Instituto Butantan. Área de concentração: Biotecnologia. Linha de pesquisa: Bioquímica, biologia molecular, espectrometria de massa. Versão do título para o português: Fisiologia molecular da digestão em Arachnida: abordagens funcional e comparativo-evolutiva. 1. Digestão 2. Aranha 3. Escorpião 4. Enzimologia 5. Proteoma 6. Transcriptoma I. Rocha, Profa. Dra. Adriana Rios Lopes I. Universidade de São Paulo. Instituto de Ciências Biomédicas. Programa de Pós-Graduação Interunidades em Biotecnologia USP/IPT/Instituto Butantan III.
    [Show full text]
  • Fasciola Gigantica
    MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF TYPE 1 CYSTATIN STEFIN-2 OF THE LIVER FLUKE FASCIOLA GIGANTICA BY MISS SINEE SIRICOON A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE DOCTOR OF PHILOSOPHY (BIOMEDICAL SCIENCES) GRADUATE PROGRAM IN BIOMEDICAL SCIENCES FACULTY OF ALLIED HEALTH SCIENCES THAMMASAT UNIVERSITY ACADEMIC YEAR 2015 COPYRIGHT OF THAMMASAT UNIVERSITY MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF TYPE 1 CYSTATIN STEFIN-2 OF THE LIVER FLUKE FASCIOLA GIGANTICA BY MISS SINEE SIRICOON A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE DOCTOR OF PHILOSOPHY (BIOMEDICAL SCIENCES) GRADUATE PROGRAM IN BIOMEDICAL SCIENCES FACULTY OF ALLIED HEALTH SCIENCES THAMMASAT UNIVERSITY ACADEMIC YEAR 2015 COPYRIGHT OF THAMMASAT UNIVERSITY (1) Dissertation Title Molecular and biochemical characterization of type 1 cystatin stefin-2 of the liver fluke Fasciola gigantica Author Miss Sinee Siricoon Degree Doctor of Philosophy (Biomedical Sciences) Department/Faculty/University Graduate Program in Biomedical Sciences Faculty of Allied Health Sciences Thammasat University Dissertation Advisor Associate Professor Hans Rudi Grams, Dr. rer. nat. Dissertation Co-Advisor Professor Vithoon Viyanant, Ph.D. Dissertation Co-Advisor Professor Peter Smooker, Ph.D. Dissertation Co-Advisor Assistant Professor Suksiri Vichasri Grams, Dr. rer. nat. Dissertation Co-Advisor Assistant Professor Amornrat Geadkaew, Ph.D. Academic Years 2015 ABSTRACT Cysteine proteases including cathepsin B and cathepsin L are involved in physiological and biological processes in all living organisms and imbalanced activity of these proteases may cause several diseases. They are also important antigens in the trematode genus Fasciola and have vital roles in parasite survival including protection, infection and nutrition of the liver fluke.
    [Show full text]
  • Investigation of the Underlying Hub Genes and Molexular Pathogensis in Gastric Cancer by Integrated Bioinformatic Analyses
    bioRxiv preprint doi: https://doi.org/10.1101/2020.12.20.423656; this version posted December 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Investigation of the underlying hub genes and molexular pathogensis in gastric cancer by integrated bioinformatic analyses Basavaraj Vastrad1, Chanabasayya Vastrad*2 1. Department of Biochemistry, Basaveshwar College of Pharmacy, Gadag, Karnataka 582103, India. 2. Biostatistics and Bioinformatics, Chanabasava Nilaya, Bharthinagar, Dharwad 580001, Karanataka, India. * Chanabasayya Vastrad [email protected] Ph: +919480073398 Chanabasava Nilaya, Bharthinagar, Dharwad 580001 , Karanataka, India bioRxiv preprint doi: https://doi.org/10.1101/2020.12.20.423656; this version posted December 22, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract The high mortality rate of gastric cancer (GC) is in part due to the absence of initial disclosure of its biomarkers. The recognition of important genes associated in GC is therefore recommended to advance clinical prognosis, diagnosis and and treatment outcomes. The current investigation used the microarray dataset GSE113255 RNA seq data from the Gene Expression Omnibus database to diagnose differentially expressed genes (DEGs). Pathway and gene ontology enrichment analyses were performed, and a proteinprotein interaction network, modules, target genes - miRNA regulatory network and target genes - TF regulatory network were constructed and analyzed. Finally, validation of hub genes was performed. The 1008 DEGs identified consisted of 505 up regulated genes and 503 down regulated genes.
    [Show full text]
  • Crystal Structure of Cathepsin X: a Flip–Flop of the Ring of His23
    st8308.qxd 03/22/2000 11:36 Page 305 Research Article 305 Crystal structure of cathepsin X: a flip–flop of the ring of His23 allows carboxy-monopeptidase and carboxy-dipeptidase activity of the protease Gregor Guncar1, Ivica Klemencic1, Boris Turk1, Vito Turk1, Adriana Karaoglanovic-Carmona2, Luiz Juliano2 and Dušan Turk1* Background: Cathepsin X is a widespread, abundantly expressed papain-like Addresses: 1Department of Biochemistry and v mammalian lysosomal cysteine protease. It exhibits carboxy-monopeptidase as Molecular Biology, Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia and 2Departamento de well as carboxy-dipeptidase activity and shares a similar activity profile with Biofisica, Escola Paulista de Medicina, Rua Tres de cathepsin B. The latter has been implicated in normal physiological events as Maio 100, 04044-020 Sao Paulo, Brazil. well as in various pathological states such as rheumatoid arthritis, Alzheimer’s disease and cancer progression. Thus the question is raised as to which of the *Corresponding author. E-mail: [email protected] two enzyme activities has actually been monitored. Key words: Alzheimer’s disease, carboxypeptidase, Results: The crystal structure of human cathepsin X has been determined at cathepsin B, cathepsin X, papain-like cysteine 2.67 Å resolution. The structure shares the common features of a papain-like protease enzyme fold, but with a unique active site. The most pronounced feature of the Received: 1 November 1999 cathepsin X structure is the mini-loop that includes a short three-residue Revisions requested: 8 December 1999 insertion protruding into the active site of the protease. The residue Tyr27 on Revisions received: 6 January 2000 one side of the loop forms the surface of the S1 substrate-binding site, and Accepted: 7 January 2000 His23 on the other side modulates both carboxy-monopeptidase as well as Published: 29 February 2000 carboxy-dipeptidase activity of the enzyme by binding the C-terminal carboxyl group of a substrate in two different sidechain conformations.
    [Show full text]
  • Deficiency for the Cysteine Protease Cathepsin L Promotes Tumor
    Oncogene (2010) 29, 1611–1621 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc ORIGINAL ARTICLE Deficiency for the cysteine protease cathepsin L promotes tumor progression in mouse epidermis J Dennema¨rker1,6, T Lohmu¨ller1,6, J Mayerle2, M Tacke1, MM Lerch2, LM Coussens3,4, C Peters1,5 and T Reinheckel1,5 1Institute for Molecular Medicine and Cell Research, Albert-Ludwigs-University Freiburg, Freiburg, Germany; 2Department of Gastroenterology, Endocrinology and Nutrition, Ernst-Moritz-Arndt University Greifswald, Greifswald, Germany; 3Department of Pathology, University of California, San Francisco, CA, USA; 4Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA and 5Ludwig Heilmeyer Comprehensive Cancer Center and Centre for Biological Signalling Studies, Albert-Ludwigs-University Freiburg, Freiburg, Germany To define a functional role for the endosomal/lysosomal Introduction cysteine protease cathepsin L (Ctsl) during squamous carcinogenesis, we generated mice harboring a constitutive Proteases have traditionally been thought to promote Ctsl deficiency in addition to epithelial expression of the invasive growth of carcinomas, contributing to the the human papillomavirus type 16 oncogenes (human spread and homing of metastasizing cancer cells (Dano cytokeratin 14 (K14)–HPV16). We found enhanced tumor et al., 1999). Proteases that function outside tumor cells progression and metastasis in the absence of Ctsl. As have been implicated in these processes because of their tumor progression in K14–HPV16 mice is dependent well-established release from tumors, causing the break- on inflammation and angiogenesis, we examined immune down of basement membranes and extracellular matrix. cell infiltration and vascularization without finding any Thus, extracellular proteases may in part facilitate effect of the Ctsl genotype.
    [Show full text]
  • Human Cathepsin V / Cathepsin L2 / Preproprotein Protein (His Tag)
    Human Cathepsin V / Cathepsin L2 / Preproprotein Protein (His Tag) Catalog Number: 10093-H08H General Information SDS-PAGE: Gene Name Synonym: CATL2; CTSL2; CTSU; CTSV; MGC125957 Protein Construction: A DNA sequence encoding the full length of human cathepsin L2 (NP_001324.2) (Met 1-Val 334) was expressed, with a C-terminal polyhistidine tag. Source: Human Expression Host: HEK293 Cells QC Testing Purity: > 95 % as determined by SDS-PAGE Bio Activity： Protein Description Measured by its ability to cleave the fluorogenic peptide substrate Z-LR- Cathepsin V (CTSV), also known as Cathepsin L2, CTSL2, and CATL2, is AMC, (R&D Systems, Catalog # ES008) . The specific activity is >1000 a member of the peptidase C1 family. It is predominantly expressed in the pmoles/min/μg. thymus and testis. Cathepsin V is also expressed in corneal epithelium, and to a lesser extent in conjuctival epithelium and skin. It is a lysosomal Endotoxin: cysteine proteinase that may play an important role in corneal physiology. It has about 75% protein sequence identity to murine cathepsin L. The fold < 1.0 EU per μg of the protein as determined by the LAL method of this enzyme is similar to the fold adopted by other members of the papain superfamily of cysteine proteases. Cathepsin V has been recently Stability: described as highly homologous to Cathepsin L and exclusively expressed Samples are stable for up to twelve months from date of receipt at -70 ℃ in human thymus and testis. Cathepsin V is the dominant cysteine protease in cortical human thymic epithelial cells, while Cathepsin L and Predicted N terminal: Val 18 Cathepsin S seem to be restricted to dendritic and macrophage-like cells.
    [Show full text]